The objective of the present work was to develop PTH (1-34)-loaded stealth nanoliposomes (PTH-LPs) by employing the use of the Quality by Design (QbD) approach. Risk identification was carried out using the Ishikawa fishbone diagram. PTH-LPs were optimized using Box Behnken Design, a type of response surface methodology to examine the effect of independent variables on dependent variables such as particle size and percentage entrapment efficiency (%EE). Design space was generated for PTH-LPs to reduce interbatch variability during the formulation development process. Furthermore, a cytotoxicity assay, cell proliferation assay, calcium calorimetric assay, mineralized nodule formation, and cellular uptake assay were carried out on MG-63 osteoblast-like cells. The results obtained from these procedures demonstrated that lipid concentration had a significant positive impact on particle size and %EE, whereas cholesterol concentration showed a reduction in %EE. The particle size and %EE of optimized formulation were found to be 147.76 ± 2.14 nm and 69.18 ± 3.62%, respectively. Optimized PTH-LPs showed the sustained release profile of the drug. In vitro cell evaluation studies showed PTH-LPs have good biocompatibility with MG-63 cells. The cell proliferation study revealed that PTH-LPs induced osteoblast differentiation which improved the formation of mineralized nodules in MG-63 cells. The outcome of the present study conclusively demonstrated the potential of the QbD concept to build quality in PTH-LPs with improved osteoanabolic therapy in osteoporosis. Osteoporosis is characterized by a progressive decline in bone mineral density ,2. It prTherefore, various drug delivery strategies have been explored to modulate PTH (1-34) release ,9,10. FoLiposomes remain as one of the most preferred drug-delivery systems ,21,22,23Several polymers are being investigated to improve the systemic circulation time of liposomes ; howeverThe development of a pharmaceutical dosage form is based on several critical aspects, including the selection of formulation variables and process parameters that can affect the quality of the final dosage form. A more systematic approach for developing a pharmaceutical formulation is Quality by Design (QbD), which is a risk- and knowledge-based quality management approach. It is “a systematic approach to development that begins with a predefined objective and emphasizes product and process understanding and process control, based on sound science and quality risk management” . RegulatIn our previous study, we screened material attributes and process parameters affecting PTH (1-34)-encapsulated lipid vesicles using Taguchi standard orthogonal array L8 design . The pre2HPO4), potassium dihydrogen phosphate (KH2PO4), acetonitrile, formic acid, and isopropyl alcohol were procured from Fischer Scientific . NBD-PE -1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium Salt), trypsin, and fetal bovine serum (FBS) were purchased from InvitrogenTM, Thermo-Fischer Scientific . Ultra-pure water from Millipore Milli-Q (Synergy UV) water purification system (Merck Millipore) was used throughout the study. All other reagents used were of analytical grade and were used without further processing. MG-63 osteoblast-like cells were procured from the National Center for Cell Science (NCCS), Pune, India. PTH (1-34), paraformaldehyde, alizarin red solution, DAPI , and cholesterol was procured from Sigma-Aldrich . Hydrogenated phosphatidylcholine (HSPC) and N-(carbonyl methoxy polyethyleneglycol-2000)-1,2-distearoylsn-glycero-3-phosphoethanolamine  were obtained as gift samples from Lipoid GmbH . Absolute ethanol was procured from Shree Chalthan Vibhag Khand, Uddyog Sahakari Mandli Ltd., Surat, India, and Sodium chloride (NaCl) from HiMedia Laboratories Pvt. Ltd. . Potassium chloride (KCl), sodium hydrogen phosphate (NaThe entire QbD-based study involves the following elements: (a) Define the Quality Targeted Product Profile (QTPP), which forms the basic design for the development of the product. QTPPs are based on knowledge space developed from the relevant scientific literature with appropriate in vivo relevance; (b) Identification of potential Critical Material Attributes (CMAs), Critical Process Parameters (CPPs), and Critical Quality Attributes (CQAs). As per the ICH Q8 R2 guideline, “CQA is a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality”; (c) Perform Risk Assessment (RA) to identify CMAs and CPPs that may have significant impact on CQAs. Among the various tools mentioned in ICH guideline Q9 for performing the RA, Failure Mode Effects Analysis (FMEA) and Failure Mode Effects and Criticality Analysis (FMECA) are the most widely used . These tIn conclusion, applying QbD in the development of a pharmaceutical product helps in better product and process understanding. Therefore, the development of PTH-LPs using the QbD approach was initiated as per guidance ICH Q8(R2) for pharmaceutical development. Further, guidance for the industry for liposomal drug products approved by the Food and Drug Administration (FDA) was also considered during the development of the liposomal formulation . Identification of QTPP and CQA  was carrAfter the experimentation and data analysis, the obtained experimental results were compared with predicted responses (CQAs/dependent variables) and the percentage residual value was calculated using the following formula;PTH-LPs were prepared by the ethanol injection method. Briefly, absolute-ethanol-containing lipid, cholesterol, and DSPE-PEG-2K were injected into 3 mL of acetate-buffer containing PTH (1-34). The resulting mixture was then stirred for a specific period and centrifuged for 60 min at 50,000 rpm. The obtained pellet was redispersed into Milli-Q water and stored at −20 °C. Malvern Zetasizer Nano ZS 90  was used to measure the particle size of optimized PTH-LPs using the photon correlation scattering (DLS) method. Prior to size assessment, the optimized PTH-LPs were diluted with Milli-Q water to achieve the proper scattering intensity. Measurements were made using 1 mL of diluted PTH-LPs in a single-use polystyrene cuvette at 25 °C with a 90° scattering angle. Zeta potential of the PTH-LPs was measured without dilution of samples at 25 °C in triplicate. v/v formic acid in Milli-Q water as an aqueous phase (A) and 0.1% v/v formic acid in acetonitrile as an organic phase (B). The injection volume was kept at 50 μL, and analysis was performed at a 210 nm wavelength [The drug content in PTH-LPs was determined using HPLC  by injecting the supernatant after ultracentrifugation of PTH-LPs at 50,000 rpm. Chromatographic separation was carried out using the XBridge BEH C18 column . The mobile phase consisted of 0.1% velength . %EE wasCryo FE-SEM  was used to inspect the morphology of optimized PTH-LPs. For this, samples were transferred on rivets mounted on a cryo-SEM sample holder. It was then submerged in liquid nitrogen for freezing. The frozen samples were fractured using a cold knife. Sublimation of samples was done for 10 min at −90 °C, and images of samples were obtained after subjecting the samples to the chamber of Cryo FE-SEM.The in vitro PTH (1-34) release from PTH-LPs was performed using a sample-separate method. Pellets of PTH-LPs were dispersed in 10 mL of PBS (pH 7.4). Then, 1 mL of liposomal dispersion was added into single microcentrifuge tubes (MCTs) for a single time point. All MCTs were incubated at 37 °C in an orbital shaker at speed of 100 rpm for 24 h. At time intervals of 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 12 h, and 24 h, MCTs were taken out of the orbital shaker and centrifuged at 50,000 rpm. The supernatant was removed, and pellets were dissolved in absolute ethanol. Samples were injected in HPLC to quantify released PTH (1-34) from PTH-LPs using a validated HPLC method.−1 of streptomycin, 100 U mL−1 of penicillin, and 10% fetal bovine serum . The cultures were maintained in T-75 flasks and incubated at 37 °C with 5% CO2.In present study, MG-63 osteoblast-like cells  were used for cellular assay. Cells were cultured in alpha-modified Eagle’s medium . The medium was supplemented with 2 mM glutamine, 100 mg mL4 cells per well in 100 μL of culture medium. The cells were incubated in an incubator at 37 °C for 24 h for attachment. After 24 h, the culture medium was removed, and cells were washed with PBS. Next, 100 μL of medium containing various concentrations of PTH (1-34) and PTH-LPs were added to a well plate and incubated for 24 h. After 24 h incubation, the medium was removed, and wells were rinsed with PBS. Then, the 100 μL medium containing 10 μL Alamar Blue reagent was added in each well and incubated for another 2 h. Absorbance was measured using a multimode UV microplate reader  at 520–590 nm. The % cell viability was obtained from the following equation:The cytotoxicity of PTH-LPs was determined by Alamar blue assay. This is a quantitative calorimetric assay based on the biochemical reduction of dye (Alamar blue) from non-fluorescent blue to fluorescent red. Briefly, MG-63 cells were seeded onto 96 well plates at density 1 × 104 cells were seeded into 48 well plates. The cells were incubated for 24 h at 37 °C. Then, the cells were treated with PTH-LPs and further incubated for 7 days. At 3, 5, and 7 days, cells were washed with PBS and treated with a 400 μL medium containing Alamar Blue reagent. After 2 h of incubation, absorbance was measured using a multimode UV microplate reader at 520–590 nm. Furthermore, light microscope images of cells treated as a control and samples on the third day of the experiment were also captured to evaluate the osteoblast proliferation potential of PTH-LPs.Cell proliferation assay was performed to evaluate the osteoblast differentiation potential of PTH-LPs on MG-36 cells. For this study, 1 × 10Free calcium concentration from medium after the 7-day incubation period of MG-63 cells was performed using a Calcium Calorimetric Assay Kit . Calcium ion concentration was measured by measuring the chromogenic complex formed between o-cresolphthalein and calcium ion. The chromogenic complex was determined spectrophotometrically by measuring the optical density at 575 nm. The optical density was proportional to the concentration of calcium ions present in the samples.The assay was performed as per manufacturer’s instructions. Briefly, 50 μL of samples or calcium standards were added to a 96-well plate. 90 μL of chromogenic reagent and 60 μL of calcium assay buffer was added to wells containing controls, samples, or standards. The assay plate was incubated for 10 min at room temperature and absorbance was measured at 575 nm using microplate reader.4 in 6-well plates and cultured in osteoblast-specific media. After the 24 h, cells were washed with PBS, and different treatments were given. Wells containing only media were considered control. After 7 days, cells were fixed with 4% paraformaldehyde for 15 min. Paraformaldehyde was removed and cells were washed with PBS. Cells were stained with Alizarin Red solution (1.5%) for 15 min. Unreacted dye was removed, and cells were rewashed 2–4 times with PBS. Then, mineral nodules were observed under a light microscope. For quantitative analysis, pyridinium chloride (10%) was added to each well to dissolve the dye, and the concentration of dye was determined by measuring the absorbance of the sample using a microplate reader at 562 nm.Alizarin Red assay was used to detect the osteoblast’s mineralized nodule formation upon treatment of the drug and PTH-LPs. For this, cells were seeded at 1 × 105) were cultured on a glass coverslip in a 35 mm culture plate for 24 h for this study. After 24 h, cells were washed with PBS and incubated with fluorescent PTH-LPs for 1, 3, and 6 h. After each time point, cells were washed with PBS and fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature. Again, cells were washed 3 times with PBS. The cell nuclei were stained with DAPI for 15 min, and excess DAPI was washed out using PBS three times. The glass coverslip was removed from the culture plate and air dried. DPX mounting agent was used for the attachment of the coverslip on the glass slide and kept at room temperature for drying. Confocal microscope analysis was performed using a laser-scanning confocal microscope . Fluorescent PTH-LPs excitation and emission wavelength were 463 nm and 536 nm respectively. Image J software was used to process the images and to perform semiquantitative analysis. Corrected Total Cell Fluorescence (CTCF) was determined to compare cellular uptake of PTH-LPs at different time points without further processing.A cellular uptake study of PTH-LPs was performed by using confocal microscopy. MG-63 cells (1 × 10n ≥ 3). Statistical significance was determined using Student’s t-test. The calculated p-values were defined as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. Nonsignificant differences are marked as n.s.The statistical analysis of the results was carried out using GraphPad Prism . All values are expressed as the mean ± standard deviation (SD)  (2009), QTPP is ‘a prospective summary of the quality characteristics of a drug product that ideally will be achieved to ensure the desired quality, taking into account of safety and efficacy of the drug product’. The present study deals with the development of pegylated nanoliposomes encapsulated with the peptide drug PTH (1-34) for systemic administration that would remain in the circulatory system for a prolonged period and deliver the drug for a longer time. Hence, the appropriate size of nanoliposomes (100–200 nm), pegylation of liposomes for longer systemic circulation, the high therapeutic concentration of the drug for therapeutic efficacy, and optimal cholesterol concentration for vesicles stability are the main QTPPs for developing the formulation. After the QTPP, the second most important step of the QbD approach is to determine crucial and potential CQAs of the drug product. CQAs are physical, chemical, biological, and microbiological parameters that should be within the defined range to determine product quality. The selection of CQAs and QTPP is based on an understanding of drug products, preliminary studies, literature review, and experience in formulation development. Risk assessment is a process to determine which CMAs and CPPs affect CQAs.Risk analysis can be done using several techniques to identify risk factors that influence CQAs. A risk assessment strategy assists in improving the quality of the process and formulation and in identifying the critical attributes that might affect the final formulation’s quality. In this study, we have utilized the Ishikawa fishbone diagram for the identification of risk factors . As per Based on the preliminary experimentation, prior experience, and risk analysis, lipid concentration, cholesterol concentration, and stirring rate were selected to optimize the PTH-LPs, whereas DSPE-MPEG-200K, stirring time, aqueous to organic ratio, and injection rate were kept constant. For determining the effects of identified CMAs and CPPs, a 3-level Box Behnken Design (BBD) with three independent factors was developed using Design-Expert software . Two different CQAs (responses/dependent factors) were evaluated; particle size and %EE. Levels of independent variables examined in this experimental design were obtained within the range obtained from preliminary experimentation and a literature search. The matrix of experimental design included 17 runs with three replicates. The layout of the experimental design with experimental runs obtained is enlisted in Various statistical modules from Design Expert software were utilized to fit the experimental data with the selected experimental design and calculate the statistical parameters. The analysis of variance (ANOVA) method was used for the calculations and data analysis of statistical parameters. Design space establishment is based on the regression models; therefore, the same software was used to generate the design space. All the QTPP criteria are met at a predetermined degree of risk. p-values less than 0.0500 indicate the model and its terms are significant. There is only a 1.42% chance that a large F-value could occur due to noise. Values greater than 0.1000 indicate that the model terms are not significant. If there are many insignificant model terms, then model reduction may improve the model. Therefore, a reduced quadratic model was used in particle size analysis to get significant model and model terms. Data obtained from ANOVA showed CMA has a significant impact on particle size, since the p-value of the model and lack of fit were 0.0142 and 0.9486, respectively. The lack of fit F-value of 0.29 implies the lack of fit is not significant relative to the pure error. Nonsignificant lack of fit is good. Furthermore, the fit’s statistical data show that the predicted R2 of 0.2234 is in reasonable agreement with the adjusted R2 of 0.3778, and the difference found between them is less than 0.2. Adequate precision is another statistical parameter that needs to be evaluated that measures the signal-to-noise ratio. A ratio greater than 4 is desirable. In the case of particle size, this ratio is 6.991, indicating an adequate signal,; hence, this model can be used to navigate the design space. Statistical data of model terms are enlisted in The lipidic vesicular size was obtained between 39.48 to 155.83 nm. Statistical analysis showed a model F-value of 5.86, and Furthermore, residual analysis was performed to find the trend of the data sets. This analysis showed that all residual plots behaved very well . Residuap-values of 0.0432 indicate model and models’ terms are significant. There is only a 4.32% chance that an F-value this large could occur due to noise. For %EE, a reduced linear model was used to obtain the significant model and model terms. The lack of fit F-value of 0.59 implies the lack of fit is not significant relative to the pure error. There is a 77.61% chance that a lack of fit F-value this large could occur due to noise. The difference between predicted R2 and adjusted R2 is less than 0.2. A ratio of 4.552 shows an adequate signal, suggesting this model is adequate for navigation of the design space. To prepare nanovesicles, lipid and cholesterol solution was injected into the drug-containing aqueous solution. Therefore, the lipid solution’s molar concentration significantly impacts the final liposomal drug concentration and %EE. %EE was found to be between 24.58% and 87.98%. Statistical analysis showed the model F-value of 4.88, and An increase in lipid concentration may increase the %EE. In the current formulation, lipid concentration had a significant role in %EE. Increased lipid concentration showed an improvement in %EE, which was depicted from all counter plots and 3D surface plots . It has Considering the target range of CQAs, a design space was generated  by consiFor the optimal formulation, the targeted CQAs were maximum entrapment of peptide into the lipid vesicles and a particle size less than 200 nm. The optimal formulation was obtained by using 60.25 ± 11.64 mM of lipid, 9.33 ± 2.57 mM of cholesterol, 3 mM of pegylated lipid, and 500 rpm stirring speed. Physicochemical characteristics such as particle size, zeta potential, and entrapment efficiency of PTH-LPs were evaluated. The particle size distribution of the optimized PTH-LPs is presented in In vitro drug release of plain PTH (1-34) and PTH (1-34) from PTH-LPs is shown in The in vitro cytotoxicity of free drug and PTH-LPs towards MG-63 cells was evaluated using Alamar Blue assay. As shown in Furthermore, as a part of the cell proliferation assay, cells were incubated for 7 days after the treatment. At 3-, 5-, and 7-day intervals, % proliferation was calculated using Alamar Blue reagent A–C. The Calcium calorimetric assay  shows thOsteoblast mineralization can be assessed by quantitative mineralization experiments such as Alizarin Red assay. Therefore, Alizarin Red assay was performed to determine the mineralization by MG-63 cells after the incubation with plain drug and PTH-LPs. Cells without treatment were considered the control. 1.5% Alizarin Red was used for staining purposes. The cellular uptake of PTH-LPs was evaluated semi quantitatively by fluorescence microscopy. The objective of the present study was to formulate PTH-LPs that could overcome the stability-related issues of the anabolic peptide. Based on the data obtained from QbD, it has been shown that this approach is the key element in the development of PTH-LPs by providing information on the CMAs and CPPs during formulation development. This approach showed that lipid concentration has a significant influence on %EE and particle size. Optimized formulation was achieved by formulating the suggested composition from software, whose quality attributes are within the specified limits. PTH-LPs showed a sustained drug release profile up to 24 h. In vitro cell experiments revealed that PTH-LPs had excellent biocompatibility with MG-63 cells. Cell proliferation and mineralized nodule formation assay suggested that PTH-LPs induced osteoblast differentiation and mineralization as well as the formation of mineralized nodules. Furthermore, the cell uptake study revealed that the PTH-LPs have a great capacity for cellular uptake in MG-63 cells. Hence, it is concluded that PTH-LPs could be a potential solution for effectively delivering PTH (1-34) in osteoporosis.


E)-1-[3-(diethoxyphosphoryl)propyl]-4-[4-(diethylamino)styryl]pyridin-1-ium bromide (ASP-PE), for mechanical mapping in live cells. ASP-PE is aggregation-induced emission active and is sensitive to the local mechanical environment. It targets the plasma membrane (PM) and intracellular mitochondria in cells by its phosphonate moiety and pyridinium. In this work, through ASP-PE staining, changes of membrane tension in the PM and mitochondria in response to varied osmotic pressure and substrate stiffness are visualized using fluorescence lifetime imaging microscopy. The mechanobiological importance of actin filaments and microtubules in the PM and mitochondria is also investigated using this probe. Computational simulations are applied to study the sensing mechanism of the probe. This study introduces a unique tool for mapping the membrane tension in the PM and mitochondria together, providing us great opportunities to study organelle’s interactions in mechanobiology.Cells are responsive to the mechanical environment, but the methods to detect simultaneously how different organelles react in mechanobiological processes remain largely unexplored. We herein report a dual organelle-targeting fluorescent probe, ( Advanced fluorescent AIE probe enables mapping of the mechanical dynamics of cell membrane and mitochondria. Mechanical forces exerted by the extracellular environment are counterbalanced by intracellular pressure and play important roles in cellular physiology and homeostasis. The impacts of these biophysical stimuli are evident in dictating cell fate and behaviors, and mechanical cues in the cellular microenvironment have been found important in many biological processes such as cancer progression (The plasma membrane (PM) is the foremost and pivotal interface that mediates cell mechanobiological responses to external mechanical stimuli  in the PM and mitochondria under external mechanical stimuli including tension, compression, and changes in substrate stiffness. Distinct mechanotype changes in the PM and mitochondria under different mechanical disturbances were revealed by ASP-PE. The role of cytoskeletons in cellular mechanobiology was also demonstrated using ASP-PE. Molecular dynamics (MD) investigation coupled to a stimulated phospholipid bilayer structure was used to investigate the relationship between membrane tension and fluorescence lifetime of the fluorescent probes in silico. Structural comparisons among ASP-PE and two styryl dyes suggested that the phosphonate moiety of ASP-PE was critical for its PM targetability and high mechanosensitivity in the target membrane.Here, we designed and synthesized a mechanosensitive dual PM-mitochondria–targeting AIE-active fluorescent probe named ASP-PE. Unlike the existing tools that track only the PM but no other intracellular compartments simultaneously, ASP-PE is a bioprobe that specifically targets both the PM and mitochondria at the same time. We demonstrated that ASP-PE could detect single-cell mechanotype from its fluorescence lifetime (τE)-N,N-diethyl-4-(2-[pyridin-4-yl)vinyl]aniline and ASP-PE were structurally characterized by 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy as well as high-resolution mass spectrometry (HRMS) (figs. S2 to S4). The photophysical properties of ASP-PE were studied and illustrated in abs) of 500 nm with a molar absorptivity (ε) of ca. 5 × 104 M−1 cm−1 and an emission peak (λem) at ca. 610 nm in ethanol solution  (The new mechanosensitive probe ASP-PE (molecular structure shown in  515 nm) . Its abs 515 nm) . In a vi 515 nm) .The cytotoxicity of ASP-PE was evaluated using the Cell Counting Kit-8 (CCK-8) assay before bioapplication. As illustrated in fig. S6, the cell viability was more than 70% at a concentration below 15 μM, demonstrating a good biocompatibility of ASP-PE in vitro. We then tested its performance in live-cell imaging by incubating HeLa cells with 10 μM ASP-PE for 15 min. ASP-PE specifically labeled the cell surface and intracellular compartments. Costaining experiments showed that these signals were colocalized with the commercial PM and the mitochondria probes  . OverallE)-4-[4-(diethylamino)styryl]-1-methylpyridin-1-ium iodide (ASP) with one methyl group locking to the pyridinium ring, which is reported as a mitochondrial probe for live-cell imaging (E)-4-[4-(diethylamino)styryl]-1-(2-octyldodecyl)pyridin-1-ium bromide (ASP-OD) with a long and branched alkyl chain on the pyridinium, which was structurally characterized by 1H- and 13C-NMR spectroscopy as well as HRMS (figs. S7 to S9). The photophysical properties of ASP and ASP-OD were depicted in the ultraviolet-visible (UV-Vis) absorption spectra (fig. S10), PL spectra (figs. S10 and S11), and time-correlated single-photon counting (TCSPC) spectra (fig. S12). These data showed that all three molecules have similar photophysical properties because of the same π-conjugation moiety. In the fluorescence imaging, ASP preferentially labeled the mitochondria in HeLa cells propyl moiety, was measured in cells (figs. S13 and S14). The mean τf of ASP in the PM and mitochondria was 4.19 and 2.65 ns, respectively, whereas the mean τf of ASP-OD in the PM and mitochondria was 1.55 and 1.28 ns, respectively. Their τf ranges or mean τf differences in the PM and mitochondria were not substantial when comparing to ASP-PE. The average ASP-PE lifetime in the PM and mitochondria were distinguishable under fluorescence lifetime imaging microscopy (FLIM) at 4.73 and 3.64 ns, respectively . In compf of ASP-PE in the PM increased significantly under hypotonic shock, but it decreased upon hypertonic treatment  of 0.9858 , causes tensile stress, whereas the hypertonic condition causes compressive pressure . The meareatment . The meaf 0.9858 . The linondition . A lineasm/liter . RegardiThe cellular microenvironment in vivo is complex and dynamic with stiffness heterogeneity that induces cellular adaptation  was used. Its mechanical property was revealed by its stress-strain curve where Young’s moduli was 855 kPa for the PDMS. In addition, the thickness of these PDMS thin films was controlled to be around 90 μm. It was thicker than the critical value of 38 μm which is the marginal thickness for cells responding to substrate stiffness . For cel surface . On the ong them . Provide. This lateral organization structurally strengthens the PM against external forces. On the other hand, microtubules are involved in maintenance of cell shape, cell adhesion, as well as organelle positioning and trafficking.The cytoskeleton is a highly dynamic protein network that provides cells with structural support, a framework for organelle movement, and a system for generation of mechanical forces. The fence and picket model proposes that actin filaments extensively tether to the PM that confines the lateral movement of molecules in the membrane f difference of ASP-PE in the PM and mitochondria under the pharmacological treatments. Under FLIM, disorganization of microtubules decreased the mean τf of ASP-PE in the PM of Noc-treated cells  and cytochalasin D (CytoD), respectively (ed cells  but not ed cells . This imed cells  but decred cells . This susn-glycero-3-phosphocholine (POPC) phospholipids, cholesterol (CHOL), and N-palmitoyl-d-erythro-sphingosylphosphorylcholine (PSM) phospholipids were first obtained by the MD simulations  at its global minimum, the Z position of ASP-PE molecule (Z = 11 Å) was closer to the phospholipid membrane center. From the global minimum to the global maximum in the phospholipid membrane center (Z = 0 Å), both the free-energy profiles of ASP and ASP-PE molecule needed to undergo a continuous endothermic process to overcome the energy barrier. For the entry of ASP-OD molecule to the phospholipid membrane, its free-energy profile first dropped to −16.20 kcal/mol and reached a minimum at Z = 12 Å. After overcoming an almost negligible energy barrier, its free-energy profile further dropped sharply until it reached the global minimum state in the phospholipid membrane center (Z = 0 Å) whose free-energy was −26.82 kcal/mol. From the simulation, it is shown that although these three molecules have the same π-conjugation moiety, they present different stable positions  and translocation abilities  in the phospholipid membrane. The calculated effective resistance coefficients and effective permeability coefficients are shown in table S1.On the basis of the sampled data from all simulation windows and the weighted histogram analysis method techniques (Z = 18 Å), it inserted vertically into the phospholipid membrane with its positively charged pyridine ring surrounded by the negatively charged phosphatidylcholine/sphingomyelin head groups. This insertion mode could enhance the electrostatic interaction between these two groups. However, when the ASP-PE molecule was in the most stable position (Z = 11 Å), it laid parallel to the phospholipid membrane with its positively charged pyridine ring was in proximity to but not surrounded by the negatively charged phosphatidylcholine/sphingomyelin head groups  of the ASP-OD molecule , their structural changes, electronic excitation properties, and reorganization energies were used to determine the fluorescence properties. Notably, different membrane tensions  were considered for each probe conformation in the phospholipid membrane during the quantitative calculation of fluorescence properties.0 state) and the first excited singlet state (S1 state) were considered. As shown in fig. S20, the root mean square deviations  between S0 state and S1 state for the ASP, ASP-PE, and ASP-OD molecules in the pure ethanol solvent were 0.067, 0.111, and 0.138 Å, respectively. When these molecules were embedded in the phospholipid membrane with a surface tension of +50 dyne/cm, the RMSDs for the ASP, ASP-PE, and ASP-OD molecules were 0.049, 0.088, and 0.087 Å, respectively. When the surface tension of phospholipid membrane was changed to 0 dyne/cm, the corresponding RMSD values decreased to 0.037, 0.067, and 0.065 Å, respectively. When the surface tension of phospholipid membrane was −200 dyne/cm, they further decreased to 0.030, 0.024, and 0.037 Å, respectively. Obviously, the phospholipid membrane could reduce the conformational fluctuations of ASP, ASP-PE, and ASP-OD molecules and increase their rigidity. The more order the phospholipid membrane is, the smaller fluctuations and more rigid of the embedded dyes will be (fig. S21).First, the structural changes between the ground state  of SConsidering that the recovery of structural rotations primarily goes through the nonradiative transition, the nonradiative decay rate constants of membrane-embedded ASP, ASP-PE, and ASP-OD molecules will decrease due to the limitation of motion. This was confirmed by the calculated nonradiative decay rate constant for ASP-PE molecule as shown in For ASP-PE molecule, when the surface tension adopted on the phospholipid membrane increased, the limitation on intramolecular motion led to a decreased nonradiative rate constant , this increases the quantum yield and fluorescence lifetime overall. Compared to the RMSD and RE values of ASP and ASP-OD, those of ASP-PE showed a higher sensitivity to the membrane tension changes, indicating that APE-PE is the best membrane tension probe among the three molecules here, which is in good agreement with the experimental results. Considering that ASP, ASP-PE, and ASP-OD molecules share the same chromophore unit, their photo-inactive moieties led to their most stable positions and postures in the membrane, which, in turn, led to their different sensitivities to the changes of membrane tension.In summary, with an electron push-pull π-conjugation skeleton and a rarely reported diethyl phosphonate moiety on the pyridinium ring, an AIE-based bioprobe ASP-PE is developed for dual PM-mitochondria–targeting and visualizing cellular mechanical properties. Referring to respective subcellular localization and biomechanical sensitivity of ASP-PE and two structurally similar styryl dyes, the structural comparisons suggest that the phosphonate ester moiety in ASP-PE acts as a mechanosensitivity enhancer in membranous organelles. In addition to sensitivity to different environments during live-cell imaging by FLIM, two distinguishable fluorescence lifetimes are exhibited in the PM and mitochondria, respectively, for ASP-PE.f changes of ASP-PE under osmotic shock and varied substrate stiffness. In hypotonic medium or on the high stiffness substrate, the cell membrane is stretched and has a high membrane tension. The probe correspondingly shows a significantly longer fluorescence lifetime. In hypertonic medium or on the low stiffness substrate, the fluorescence lifetimes of ASP-PE in the cell membrane are much shorter. The signal of ASP-PE indicates that the mechanical properties of mitochondria are almost unaffected by substrate stiffness as the PM does. While under osmotic stress, the mitochondria have altered fluorescence lifetime signals with the same trend as the PM. It demonstrates that the probe can be used in studying the relation of the PM and mitochondria.In high-tension membrane, ASP-PE experiences less freedom of molecular motions and exhibits brighter emission and longer fluorescence lifetime due to the RIM effect. The fluorescence lifetime of ASP-PE in the cell membrane of HeLa cells is linearly correlated with the osmotic pressure in the medium. With the dual targeting feature of ASP-PE, changes of mechanical properties not only in the PM but also in intracellular mitochondria are studied. Different mechanobiological responses in these membrane systems are revealed by the τFurthermore, ASP-PE staining under pharmacological treatments reveals that the PM and mitochondrial membranes are distinctively influenced by microtubules or actin disorganization. This further implies that PM and mitochondrial membranes encounter respective challenges during mechanical stress. Meanwhile, it should be noted that cell contraction or cell volume changes accompanied may alter the focal planes of the PM and mitochondria differently in adherent cells. In those scenarios, it may be challenging to unmix the signals from the PM and mitochondria under the wide-field FLIM. In addition to the experimental data, in silico investigation is used to understand and verify the hypothesis that correlates the membrane tension of PM (or mitochondria) and the fluorescence lifetime of the dual organelle targeting mechanosensitive probe ASP-PE. The computational analysis reveals that the non-π-conjugated units present in the pyridine ring in these three molecules greatly affect their most favorite positions and binding postures in the membrane, which further led to different sensitivities to membrane tension changes.Together, the promising sensitivity of ASP-PE to mechanical stress and mechanobiological events demonstrates a rapid and reliable approach for exploring cellular mechanosensing. Its potential application in mechanobiological studies can further be widened by modifying this mechanosensitive bioprobe with organelle-targeting signals to reveal organelle mechanosensitivity in real time during dynamic processes, such as morphogenesis and cell migration, with the aid of FLIM.All chemical reactions were performed under an inert nitrogen atmosphere with the use of a Schlenk line. Glassware was dried in an oven before use. Commercially available reagents were used without purification. All the reagents for chemical synthesis were purchased from Tokyo Chemical Industry Co. Ltd., Sigma-Aldrich, Acros Organics, or J&K Scientific. Dry solvents were purchased from the abovementioned companies and stored in the presence of activated 3-Å molecular sieves. All the reactions were monitored by thin-layer chromatography with Merck precoated aluminum plates. Products were purified by column chromatography on silica gel (230 to 400 mesh from Merck or Davisil LC60Å 40 to 63 μm from W. R. Grace & Co.-Conn). The CCK-8 from Yeasen Biotech was used for testing cytotoxicity. The commercially available probes used were BioTracker 405 Blue Mitochondria Dye (Sigma-Aldrich) and CellMask Deep Red Plasma Membrane Stain (Thermo Fisher Scientific). PDMS kits of SYLGARD184 were purchased from Dow Corning. Gelatin, CytoD, and Noc were bought from Sigma-Aldrich.3 on a Bruker AVANCE III 500 (or 400) MHz NMR spectrometer, and tetramethylsilane was exploited as an internal standard for calibrating the chemical shift. Electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) MS was performed on an Agilent 6540 liquid chromatography–ESI-Q-TOF mass spectrometer. UV-Vis absorption spectroscopy was performed on a Molecular Devices SpectraMax M2e in different solutions at 293 K. The solution emission spectra and TCSPC of the chemical probes were measured on a PerkinElmer fluorescence spectrometer LS 55 and FLS1000 PL spectrometer at 293 K, respectively. The fluorescence decay curves were analyzed using an algorithm fitting routine supplied by Fluoracle and were shown to follow a biexponential function.Proton and carbon NMR spectra were measured in CDCl2. Unless specified otherwise, cells were seeded on a gelatin-coated 35-mm glass-bottom cell culture dish (NEST) at least 24 hours before the experiments. The fluorescence staining was performed by incubating the cells with 10 μM ASP-PE, ASP, or ASP-OD at 37°C for 15 min and then washed with phosphate-buffered saline (PBS) for three times before imaging. For the osmotic shock, HeLa cells were stained with the probe, followed by replacing the culture medium with solutions with different osmolarities. Fluorescence lifetime imaging was performed immediately after osmotic shock. Hepes-buffered DMEM  with ~360 mOsm/liter was treated as isotonic medium. The hypotonic medium (75 and 162 mOsm/liter) were prepared by diluting the Hepes-buffered DMEM with Milli-Q water, whereas the hypertonic medium (482 and 750 mOsm/liter) were prepared by adding NaCl into the Hepes-buffered DMEM.HeLa cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% (v/v) fetal bovine serum (Gibco) and 1% (v/v) penicillin-streptomycin and were maintained in a humidified incubator at 37°C in the presence of 5% COConfocal imaging was performed using a laser scanning confocal microscope (Zeiss LSM 880) equipped with a Plan-Apochromat 63×/1.40–numerical aperture (NA) objective lens, a photomultiplier tube, and a Gallium arsenide phosphide detector controlled by ZEN software (Carl Zeiss). The excitation lasers and the band-pass filters used were as follows: a 514-nm argon laser and an emission filter of 530 to 630 nm for ASP-PE, ASP, and ASP-OD; a 405-nm diode laser and an emission filter of 410 to 490 nm for BioTracker 405 Blue Mitochondria Dye; and a 633-nm HeNe laser and an emission filter of 655 to 705 nm for CellMask Deep Red Plasma Membrane Stain. Digital images were acquired and pseudo-colored with ZEN software (ZEN 2.5 lite). The degree of colocalization was evaluated by the plug-in JACoP in ImageJ .ex = 515 nm; pco.flim laser, PCO AG, Germany) with a specified power at 100 mW, and a filter cube from Nikon containing an excitation filter (514/20 nm) and a long-pass emission filter (525 nm). Before the experiment, a reference of a standard sample slide with a known lifetime of 3.55 ns  was measured. Throughout all the experiments, the laser power was set to 10% via Omicron Control Center  with the exposure time at 100 ms and the modulation frequency at 30 MHz controlled using NIS-Elements AR  integrated with a module for the pco.flim camera. By measuring the phase shift acquired from photons emitted induced by the sinusoidally modulated laser light source, the lifetime map of the interest fields and the lifetime distributions were collected and analyzed using NIS-Elements AR .The FLIM core system consisted of a wide-field complementary metal oxide semiconductor frequency-domain FLIM camera , an inverted fluorescence microscope  equipped with a Plan-Apochromat Lambda 60×/1.4-NA objective lens, a homogeneously illuminating laser light source . A blend with 10:1  of SYLGARD 184 PDMS was prepared by stirring. For live-cell imaging, the construction of a PDMS well-included two parts, which were a thin-bottom layer and a frame structure. All the PDMS mixtures were vacuum-degassed in a desiccator for 1 hour. Then, the thin-bottom layer was prepared by the float-on-water method  in fresh culture medium, and DMSO was used as the vehicle control. After incubating the cells with the above solutions for 24 hours, 10 μl of CCK-8 stock solution in PBS was added to each well, and cells were incubated for an additional 4 hours. The absorbance was measured using a SpectraMax M2e microplate reader (Molecular Devices) at a wavelength of 450 nm, and the cell viability percentage was calculated from the optical density.The cytotoxicity was evaluated using a standard CCK-8. Briefly, HeLa cells were seeded in a 96-well plate at a density of 7 × 10The tensile bar strips of the PDMS substrates were prepared by casting the PDMS blends in a mold with shapes in compliance with ASTM D412 Type C and curing at 75°C for 1 hour. Testing was done on Instron 5942 Micro Newton Tester . The samples were stretched at a rate of 50 mm/min until failure, following ASTM D412. The Young’s modulus of the PDMS strips was subsequently determined automatically by the in-house software provided by Instron in which the steepest slope, which is the Young’s modulus, was calculated on the initial linear portion of the curve using least-square fit on the test data.t test or one-way analysis of variance (ANOVA) with Tukey’s post hoc test. A P value less than 0.05 was considered statistically significant, and n is the number of independent experiments.Statistical significance was determined using two-tailed unpaired Detailed computational methods, including (i) the preparation of ternary component–based phospholipid bilayer model, (ii) MD modeling, (iii) pulling MD simulation, (iv) free-energy profiles simulation, (v) permeability coefficient calculations, and (vi) quantum mechanics/molecular mechanics calculations are fully described in the Supplementary Materials.


Catechin possesses a potential anti-inflammatory activity, but its anti-inflammatory mechanism is still unclear. Herein, the analysis of network pharmacology showed that catechin might mediate ferroptosis on macrophages to exhibit a significant anti-inflammatory effect on RAW264.7. The metabolomics further indicated that catechin might influence ferroptosis by activating two pathways of cysteine and methionine metabolism and glutathione metabolism, and inhibiting the pathway of ferroptosis to promote the reduction of l-methionine-s-oxide and s-glutathionyl-l-cysteine, and the reduction and synthesis of γ-glutamylcysteine. Furthermore, related proteins  in three metabolic pathways and ferroptosis-related proteins (GPX4 and SLC7A11) might be relevant to catechin through molecular docking. Thus, we speculate that catechin plays an anti-inflammatory effect through mediating ferroptosis on RAW264.7, which still needs further focus on the detailed molecular mechanism. Inflammation is an adaptive response of the body to harmful stimuli produced by the outside world or internal organism, such as infection and tissue injury . It playEugenia brasiliensis Lam. (Myrtaceae) leaves has an anti-inflammatory activity due to the inhibition of the migration of inflammatory mediators. As a result, they speculated that this effect might be related to catechin and gallic catechin contained in ECW [2 and catechin are better than the inhibitory effects of their single use on acrylamide. Acrylamide is a carcinogenic and toxic substance and will cause oxidative damage and systemic inflammation [Catechin belongs to the flavonoid family which is also known as flavan-3-ols (or flavanols). It is a natural polyphenol compound that mainly exists in green tea, red wine, strawberries, black grapes and apricots . The grod in ECW . In addiammation ,19. It iammation . HoweverIn this study, we preliminarily verified the cytotoxicity and anti-inflammatory activity of catechin. Secondly, the analysis of network pharmacology and metabolomics were integrated to clarify the anti-inflammatory capability of catechin mediated ferroptosis on RAW264.7 according to the results of obtained differential metabolites and related metabolic pathways. Finally, we constructed the binding modes of molecular docking of catechin and related proteins to verify their structure–activity relationships.Catechin was purchased from Solarbio Science & Technology Co.LTD . Lipopolysaccharide (LPS) was purchased from Yuanye Bio-Technology Co.LTD . Macrophage RAW264.7 cells stored in liquid nitrogen were purchased from Jinan University . Dulbecco Modified Eagle Medium (DMEM) was purchased from Thermo Fisher Scientific Inc . Phosphate buffer solution (PBS), penicillin-streptomycin and fetal bovine serum (FBS) were supplied by Gibco Life Technologies . Cell Counting Kit-8 (CCK-8) whose product code was CK04 was purchased from Dongren Chemical Technology Co. LTD . Nitric oxide (NO) kit whose product code was S0021S was purchased from Beyotime Biotechnology Co. LTD . Aspirin was purchased from Solarbio Science & Technology Co.LTD . Other mentioned chemical materials were all purchased from Guangzhou Chemical Co. LTD . All other reagents used in our research were of analytical grade.2, 37 °C) to culture macrophage RAW264.7 cells which were grown and reproduced in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin. We continued further experiments when the sterile tissue culture flasks were filled with cells.The detailed steps of cell culture have been reported . We offe2, 37 °C for 24 h before 100 μL/well of them were adjusted in 96-well plates at a density of 3.0 × 104 cells/mL. Then, 10 μL of catechin was added at the final concentrations of 0, 10, 20, 40 and 80 μg/mL . After another 24 h of incubation, the solution in each well was discarded, and 100 μL of DMEM and 10 μL of CCK-8 solution were added per well. The absorbance of the cultured samples was recorded using a microplate reader at 450 nm after 2 h incubation.The evaluation of the cell viability of macrophage RAW264.7 cells used a CCK-8 assay. The cells were hatched in a humidified atmosphere with 5% CO4 cells/mL in 24-well plates were cultured for 24 h at 37 °C. Then, the supernatant was discarded. Meanwhile, 50 μL of catechin at the concentrations of 0, 10, 20 and 40 μg/mL and 50 μL of aspirin (40 μg/mL) were added per well after the addition of 50 μL of LPS (2.5 μg/mL) . Aspirin, one of the anti-inflammatory drugs, is the positive control group [The NO assay of RAW264.7 cells through using commercial Kits could evaluate anti-inflammatory activity. First, 1 mL/well of RAW264.7 cells with a density of 1.0 × 10ol group . After aNetwork pharmacology can be applied to preliminarily predict the potential targets, pathways and mechanisms of the anti-inflammatory activity of bioactive components . Detaile7 cell/plate. The positive control group of LPS samples was added at the concentrations of 2.5 μg/mL for 24 h. Compared with the positive control group, the model group of catechin samples was added at the concentrations of 20 μg/mL for 24 h . Subsequently, we added the mixture  to the cells, and then using ultrasonication homogenized the samples for 30 min at 4 °C. The samples were centrifuged for 15 min  and placed for 1 h at −20 °C to remove the protein. The supernatant was collected and then dried in a vacuum centrifuge. Before the LC−MS analysis, samples were dissolved in acetonitrile/water  solvent. Data acquisition and analysis were performed with XCMS software . In order to normalize the acquired metabolites, pareto-scaled principal component analysis (PCA), partial least-square discriminant analysis (PLS-DA) and orthogonal partial least-square discriminant analysis (OPLS-DA) were performed [p-value produced by OPLS-DA were applied to discover the contributable variable for classification.The metabolomic analysis on macrophage RAW264.7 cells was determined via a standard metabolic operating procedure . We set erformed . The folThe specific methods of molecular docking have been reported . Firstlyt-test with SPSS 20.0 to compare the control and treatment groups; p < 0.05 was considered as statistically significant.Results were represented as the means ± SD for each experimental group. The data were analyzed by one-way ANOVA followed by a Student’s p < 0.0001) which was about three times that of the control group. It indicated that cell inflammation was successfully induced. Compared with the LPS group, the production of NO had a significant decrease (p < 0.0001) after the addition of catechin. Meanwhile, the production of NO also had a significant decrease (p < 0.0001) after the addition of aspirin (40 μg/mL). Thus, we concluded that catechin had an obvious effect on stimulating macrophages to release NO in a dose-dependent manner (10 μg/mL–40 μg/mL) within the using concentration range. These results were also consistent with the existing research [In our study, we measured the cytotoxicity of catechin on macrophage RAW264.7 cells by using the CCK-8 kit A. Comparresearch .p-value under 0.006 are shown in the Sankey diagram  which belongs to the methionine sulphoxide reductase . L-methiWe verified whether catechin interacted with related proteins  in three metabolic pathways and ferroptosis-related proteins (SLC7A11 and GPX4) in the anti-inflammatory process by molecular docking. The results showed that all related proteins had a strong affinity with catechin and the combinations were presented by their 3D and 2D structures . MeanwhiBased on the above results, we hypothesized the ferroptosis-mediated anti-inflammatory effect of catechin. We further speculated that catechin could activate two metabolic pathways  and inhibit the pathway of ferroptosis. This might achieve an anti-inflammatory effect by exerting their synergistic effects in inflammatory diseases to prevent or delay the occurrence of ferroptosis.As we all know, cells in the body’s innate immune system will produce a large number of ROS such as superoxide and hydrogen peroxide when inflammation occurs. ROS can eliminate pathogens that can cause inflammatory factors in the body . It can S-glutathionyl-l-cysteine, another metabolite in the pathway of cysteine and methionine, may also play an important role in ferroptosis mediating the anti-inflammatory progress of catechin. L-cysteine will be glutathionated to s-glutathionyl-l-cysteine to prevent the occurrence of the irreversible oxidation of l-cysteine under the inflammation environment with a great quantity of ROS. Based on the expression changes of this metabolite in the different groups, we speculated that catechin could activate the activity of CDR to promote the reduction of s-glutathionyl-l-cysteine to l-cysteine.The differential expression of some enzymes in cells such as SLC7A11 can lead to inflammation. SLC7A11 mainly transports amino acids and is also related to the balance of oxidative stress ,56. SomeIn addition, the results showed that catechin might also activate the glutathione metabolic pathway. The increasing level of ROS can lead to γ-glutamylcysteine being oxidized to bis-γ-glutamylcysteine under the inflammatory environment. Then, the decrease of γ-glutamylcysteine within the intracellular will cause the influence on the regulation of ferroptosis. Thus, we speculated that catechin could activate the glutathione metabolic pathway to arouse the GSR to promote the bis-γ-glutamylcysteine reduction. It might be an effective way to regulate the ferroptosis in time in the inflammatory environment to avoid the further deterioration of inflammation and achieve the purpose of anti-inflammation.Collectively, we mapped the molecular mechanism of the anti-inflammation of catechin mediated ferroptosis on RAW264.7, as shown in In summary, after the preliminary identification of the anti-inflammatory activity (NO reduction) of catechin on macrophage RAW264.7 cells, we found that ferroptosis might be involved in the anti-inflammatory process of catechin through network pharmacology analysis. Further, three metabolic pathways  and three differential metabolites  closely related to ferroptosis were obtained through metabolomic analysis. Meanwhile, the results of molecular docking also showed that catechin was highly associated with related proteins  in three metabolic pathways and ferroptosis-related proteins (GPX4 and SLC7A11). In addition, we also focused on the potential mechanism of catechin mediating ferroptosis to exert an anti-inflammatory effect on RAW264.7 cells. In the future, we need to conduct more experiments such as constructing animal models to further clarify and verify the specific mechanism of anti-inflammatory activity of catechin mediating ferroptosis. This could provide more reliable results for the development of catechin as a new anti-inflammatory drug.


Lonicera caerulea L.) is an oriental herbal medicine reported to have beneficial effects on metabolic disorders, such as obesity and non-alcoholic fatty liver disease. The fruit part of HB is rich in anthocyanin, a type of polyphenol. Most studies credit the antioxidant and anti-inflammatory properties of HB as the mechanisms of its effectiveness. This study investigated the inhibitory effects of HB on lipase using an in vitro assay and the modulatory effect of HB on gut microbiota in high-fat diet (HFD)-fed mice. HB inhibited pancreatic lipase activity with IC50 values of approximately 0.47 mg/mL. The fecal triglyceride (TG) levels were higher from the HFD of the HB-fed mice than they were for the control mice. Moreover, the fecal microbiota from the HFD of the HB-fed mice had relatively lower Firmicutes and higher Bacteroidetes than that from the HFD-only mice. These results suggest that HB modulates gut microbiota composition, which may contribute to body fat reduction. Hence, HB could present a useful agent for treating metabolic diseases through lower TG uptake and the regulation of gut microflora.Honeysuckle berry (HB,  Obesity is caused by the overconsumption of food and insufficient energy expenditure, and the prevalence of obesity is increasing worldwide ,2. ObesiLipid metabolism is essential for controlling obesity, but there is another mechanism of obesity treatment from a different perspective. One way to reduce the extra energy that is synthesized as body fat is to interfere with the digestion of fat or make it difficult for its absorption in the small intestine .Streptomyces toxitricini and blocks the absorption of lipids to inhibit lipid accumulation as described above [Orlistat  is the best-known pancreatic lipase inhibitor currently used to treat obesity ,10. Orlied above . On the ed above . In addied above .Firmicutes/Bacteroidetes ratio compared to their normal-weight counterparts [Dysbiosis causes systemic inflammation that is associated with a higher risk of obesity . The gutterparts .Lonicera caerulea L.) is used as a traditional medicine because of its antioxidant and anti-inflammatory properties [Honeysuckle berry  staining of epididymal fat were performed at the end of the experiment A. VisualThe adipogenesis and lipogenesis regulator genes were examined to determine the effects of HB on the lipid metabolism in adipose tissue. For adipogenesis, mRNA expressions of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and fatty acid-binding protein 4 (FABP4) in the HC group were significantly higher than in the NC group. The PPARγ, C/EBPα, and FABP4 mRNA expression levels were significantly lower in the HB group than in the HC group A–C. For The levels of Uncoupling protein 1 (UCP-1) and PPARγ coactivator 1-alpha (PGC1α) expression were quantified by qRT-PCR to evaluate the effects of HB on thermogenesis in the adipose tissue. There was a significant up-regulation of the mRNA expression of UCP-1 in the HB400 group compared to the NC, PC, HB100, and HB200, as well as the HC group. In addition, the mRNA expression of PGC1α tends to be upregulated in the HB100, HB200, and HB400 groups compared to that in the HC group, but there was no significant difference A,B.Firmicutes and Bacteroidetes in the HC group was significantly different from the NC group and the HB200 group  caused by the HFD [Lachnospiraceae NK4A136 group and RuminococcaceaeUCG-014 group, which produce butyrate, were found to be enriched in the HFD-fed mice treated with an anti-obesity agent [The role of the gut microbiota in homeostasis, metabolism, adiposity, and energy regulation, as well as in vitamin production and nutrient harvesting, which together are related to obesity and obesity-associated disorders, continues to be investigated ,38. Anthes ratio ,41. This the HFD . Also, tty agent ,44. In tty agent ,44, suggRaw HB fruits were extracted as described previously . The amoThe substrate p-nitrophenyl butyrate (p-NPB) was used to perform the pancreatic lipase inhibition assay and the method was slightly modified from the previous assay method . BrieflyForty-eight six-week-old male C57BL/6 mice were purchased from Orient Bio Co. Ltd.  and maintained in a temperature and humidity-controlled room on a 12 h/12 h light/dark cycle at Gachon University, Korea. They were given access to food and drinking water ad libitum over a one-week acclimation period. The mice were randomly split into six groups of eight mice each: NC , HC , PC , HB100 (HFD with HB100 mg/kg), HB200 (HFD with HB200 mg/kg), and HB400 (HFD with HB400 mg/kg). The experimental extracts were dissolved in a 0.5% aqueous solution of CMC-Na . Orlistat and different doses of HB were administered orally to PC and the experimental group mice, respectively, whereas the solvent was administered orally to the NC and HC group mice. For eight weeks, oral administration was performed once per day. All the animals were cared for, operated in compliance with Gachon University’s guidelines for the care and use of laboratory animals (reference number: GIACUC-R2020009).2. The epididymal fat tissue was dissected and weighed immediately. The feces of the mice were collected the day before the end of the experiment. All samples were stored at −80 °C until analysis.After 12 h of fasting, the mice were euthanized with COAfter drying for 24 h at 60 °C in a drying oven , 250 mg of fecal samples were homogenized with 2 mL of phosphate-buffered saline. Fecal fat was extracted using the method reported by Folch et al. . The fecH&E staining was performed according to the previously described method . BrieflyThe total RNA was extracted from the epididymal fat tissue, and the cDNA was synthesized from 50 ng/μL RNA. qRT-PCR was performed using an ABI QuantStudio 3 . http://huttenhower.sph.harvard.edu/galaxy/, accessed on 11 May 2022) was performed to compare the taxon microbes between HC and HB200 groups.Fecal microbiota analysis was performed pursuant to a previous study with some modifications . Brieflyt-test, using SPSS 25 , was performed for two group comparisons. p values < 0.05 were considered significant.Statistical differences of all data were analyzed using Duncan’s multiple range test after one-way analysis of the variance. Student’s Overall, this study showed that HB significantly inhibits pancreatic lipase activity, reducing dietary fat absorption. The anti-obesity efficacy of HB was comparable to that of the positive control, orlistat. Moreover, HB positively modulates the gut microbiome. These findings provide scientific evidence that HB prevents obesity and modulates the gut microbiota in HFD-induced obese mice.


Our study showed that A1 mismatch was not associated with worse outcomes at one year post-HTx.There are subtypes within blood type A, termed non-A1, that have reduced expression of A antigen on cell surfaces. This can result in the development of anti-A1 antibodies. There is limited information regarding the impact of this in heart transplant (HTx) recipients. We conducted a single-center cohort study of 142 Type A HTx recipients in which we compared outcomes of a match group (an A1/O heart into an A1 recipient or a non-A1/O heart into a non-A1 recipient) with a mismatch group (an A1 heart into a non-A1 recipient or a non-A1 heart into an A1 recipient). At one year post-transplant, there were no differences between the groups in survival, freedom from non-fatal major adverse cardiovascular events, freedom from any treated rejection, or freedom from cardiac allograft vasculopathy. There was an increased hospital length of stay in the mismatch group (13.5 vs. 17.1 days,  ABO blood type compatibility is a prerequisite for heart transplantation (HTx) due to the risk of hyperacute rejection mediated by ABO antibodies . These aWithin blood type A there are subtypes, termed non-A1, which have reduced antigen expression and density because of variation in transferase specificity and efficiency. For example, the A2 subgroup, which makes up 20% of group A individuals, expresses fewer A antigen epitopes per red blood cell and is considered less immunogenic .Research into the safety of organ transplantation across the A1 barrier has been very limited. There are a few case reports of non-A1 recipients with anti-A1 titers of ≤ 1:8 receiving A1 kidneys that did not result in allograft rejection ,5,6. A rThis was a single-center cohort study conducted at Cedars-Sinai Medical Center in Los Angeles, California. Adults aged 18 years or older with blood type A who received a HTx between 2013 and 2020 were eligible for inclusion in the study. Subjects were excluded if they had a previous HTx or if they were younger than 18 years old. Subjects’ blood samples were collected and tested for the A1 subtype if there was a discrepancy in blood type testing. Anti-A1 antibody titer testing was performed once, at the time of enrollment, on all subjects who were identified as non-A1. Donor blood types (A versus O) and A subtypes (A1 versus non-A1) were collected from the United Network of Organ Sharing (UNOS).Desensitization therapy was performed if calculated panel reactive antibodies (cPRA) were greater than 50–70%. All subjects received a standard post-HTx immunosuppression regimen of prednisone, mycophenolate mofetil, and tacrolimus. Blood subtype and the presence/absence of anti-A1 antibodies did not influence desensitization or immunosuppression strategy. Surveillance for rejection via endomyocardial biopsy was performed at protocolized intervals for all subjects regardless of A1 matching. Acute cellular rejection was defined according to the International Society for Heart and Lung Transplantation (ISHLT) 2004 grading system . AntibodAll other demographic and clinical information was collected from chart review utilizing the Cedars-Sinai Electronic Medical Record (EPIC™). To account for the 2018 change in status listings, we defined “urgent status at transplant” as status 1A in the previous scheme and statuses 1, 2, and 3 in the current scheme. Predicted heart mass (PHM) was calculated using a UNOS calculator .Our outcomes of interest were survival, freedom from CAV, freedom from non-fatal major adverse cardiac events , percutaneous coronary intervention (PCI), defibrillator/pacemaker implant (ICD/PM), or stroke), and freedom from any treated rejection (ATR), acute cellular rejection (ACR), and antibody-mediated rejection (AMR) at one year post-HTx. We assessed subclinical markers, including ejection fraction (EF) and donor-specific antibodies (DSA), at one year. We also collected immediate post-HTx data including primary graft dysfunction (PGD), vasoplegia, and length of stay (LOS) in the intensive care unit (ICU) and hospital.p-values < 0.05 were considered significant. Statistical analysis was performed using the data analysis program, SPSS version 24.0 .Results were compared between a “match” group (an A1/O donor into an A1 recipient or a non-A1/O donor into a non-A1 recipient) and a “mismatch” group (an A1 donor into a non-A1 recipient or a non-A1 donor into an A1 recipient). Continuous variables were reported as mean ± standard deviation and compared using the independent samples t-test. Categorical variables were reported as percentages and compared using Fischer’s exact test. Survival was calculated using the Kaplan–Meier method. All comparisons were two-tailed, and The study protocol was approved by the Cedars-Sinai institutional review board .We identified 150 patients with blood type A who were transplanted between 2013 and 2020. Of 142 enrolled subjects, 8 were excluded because they had a previous HTx. Subjects were enrolled between 10 days and 6.5 years after HTx. with an average of 2.4 years between HTx and enrollment. Fifty-five (39%) subjects were enrolled within one year post-transplant. Allocation into study groups is depicted in p = 0.02). All other baseline demographics, including recipient age, donor age, sex, body mass index, and predicted heart mass, were similar between groups. There were no differences in comorbidities such as diabetes, hypertension, or prior cardiac surgery. Both groups had similar types of underlying cardiomyopathy. The two groups had similar risks for pre-transplant sensitization, as indicated by rates of previous pregnancy, previous blood transfusions, PRA with mean fluorescence intensity (MFI) > 5000, pre-transplant desensitization, and induction with anti-thymocyte globulin (ATG). Subjects in the match and mismatch groups had similar waitlist trajectories with no significant difference in time spent on the waitlist. Each group had similar proportions of subjects who were seriously ill, as represented by an urgent transplant status, and had similar kidney function, as measured via creatinine immediately prior to transplant. Ischemic time in the donor heart was similar between groups.Pre-transplant clinical characteristics are shown in p = 0.58), freedom from CAV , and freedom from NF-MACE . There were seven NF-MACE events in the match group: one HF, one PCI, one ICD/PM, and four strokes. There were three NF-MACE events in the mismatch group: two ICD/PMs and one stroke. The mismatch group did not have an increased incidence of immune-mediated adverse events, with similar rates of freedom from ATR , freedom from ACR , and freedom from AMR  between groups. At a subclinical level, there was no difference in ejection fraction  or the incidence of donor-specific antibodies  at one year.Post-transplant outcomes are shown in p = 0.04), but not in the ICU . This may have been driven by numerically higher rates of PGD  and vasoplegia , although these differences were not statistically significant.The mismatch group did have a higher post-transplant LOS in the hospital overall (13.5 vs. 17.1 days, In this study, we divided HTx recipients with blood type A into match and mismatch groups based on the donor and recipient subtypes. These two groups were very similar in terms of demographics, comorbidities, severity of illness while on the transplant waitlist, and risk for sensitization. We did not observe any significant difference in one-year outcomes between these two groups.The mismatch group was further broken down into two subgroups. For A1 recipients who received non-A1 hearts, we expected comparable outcomes with the match group given there were no foreign antigens to sensitize the recipient. However, for non-A1 recipients, the presence of A1 antigen on the donor heart may have resulted in the development of antibodies and subsequent adverse outcomes. We also expected subclinical evidence of an immune response in the form of DSA, which is a major risk factor for the development of AMR and CAV and a poor prognostic factor for mortality . AlthougWe did find that the mismatch group had a higher hospital LOS after their HTx. This may have been driven by a higher incidence of PGD and vasoplegia. The mechanisms underlying PGD and vasoplegia are poorly defined; extensive research has revealed various donor, recipient, and intraoperative risk factors, although none of these are related to blood type mismatch ,13. It iEven though they had a higher hospital LOS, the mismatch group had similar one-year outcomes compared to the match group. Our study offers preliminary evidence that suggests A1-mismatched HTx can be performed safely. If future studies confirm our results, donor hearts can be more efficiently allocated. Currently, non-A1 recipients with anti-A1 antibodies often receive O organs ; this caAnother way to take advantage of the reduced immunogenicity of non-A1 subtypes is transplanting these organs across the ABO barrier . This prAn important caveat is that no study of A1-mismatched organ transplantation has found recipient anti-A1 antibody titers >1:8, including ours. Anti-A1 antibodies are rare, with a prevalence of 1% in A2 individuals. When they do exist, they are often non-reactive at body temperature and considered clinically insignificant . Our stuGiven the increased LOS in the mismatch group, further study is needed to determine whether these associations exist. Such a study should include daily titer monitoring in the peri-HTx period as well as during episodes of suspected rejection. This would provide valuable information on the trajectory of anti-A1 antibody titers in response to HTx and its associated immunosuppression. Previous studies lacked consistency in checking titers before and/or after transplant and have not been able to provide this level of detail. Additional information regarding early post-transplant biomarkers, such as troponin and B-type natriuretic peptide, would also be useful. Rather than emphasizing A subtyping, more frequent titer monitoring may allow for its use in the prognostication of HTx outcomes, and potentially influence decisions on whether to perform HTx or modify immunosuppression.A limitation of this study is that with an average of 2.3 years between HTx and enrollment, only 39% of our subjects were enrolled prior to the one-year mark. Subjects who had short-term post-HTx morbidity and mortality were therefore likely under-represented, resulting in reported outcomes that are better than expected. Although this effect would have had an equal impact on the match and mismatch groups, our study likely minimizes any possible effect that A1 mismatch may have had on adverse outcomes in the early post-HTx period. The increased LOS in the mismatch group already suggests higher short-term risk in these transplants. A fully prospective study with enrollment of all subjects prior to HTx is required to provide more data regarding early adverse events.As we did not collect data beyond one year post-HTx, we were unable to fully assess the incidence of CAV, which increases with time and affects up to 50% of recipients at ten years post-HTx . It is cOur study is the largest analysis of A1-mismatched HTx to date. Although it was limited by delayed enrollment of subjects and short follow-up time, our results provide an encouraging initial sign that A1-mismatched HTx does not result in worse outcomes at one year. Future study focused specifically on A1 into non-A1 HTx with a large cohort entirely enrolled prior to HTx and a longer follow-up interval is required to confirm the safety of A1-mismatched HTx. Emphasis should be placed on examining the increased hospital LOS that was found in our mismatch group. If future studies establish the safety of A1-mismatched HTx, elimination of the A1 barrier would reduce waitlist time, especially for non-A1 and O recipients.


Oral delivery is the most convenient drug administration route. However, oral delivery of peptides is extremely challenging due to the physical and chemical barriers within the gastrointestinal tract. Polysaccharides are often utilized as polymeric biomaterials in drug delivery. Among these, dietary polysaccharides extracted from okra, yam, and spirulina have been reported to stimulate innate immunity with well-known nutritional benefits. In this study, we developed a dietary-polysaccharide-modified fish-oil-based emulsion for oral co-delivery of a hydrophilic PD-L1 blocking peptide and the hydrophobic small molecule simvastatin. The optimal emulsion was nano-sized and exhibited a negative surface charge, high drug encapsulation efficiency of over 97%, low viscosity, and sustained drug release manner. The formulation could significantly increase the uptake of peptides by intestinal Caco-2 cells, which demonstrated the great potential of the formulation for promoting the oral absorption of peptides. Additionally, these dietary polysaccharides could promote dendritic cell maturation and cytokine expression in macrophages, demonstrating that these nutraceutical polysaccharides had dual roles of functioning as promising colloidal delivery systems and as potential immune modulators or adjuvants. Thus, this food-based colloidal delivery system shows promise for the oral delivery of peptide drugs and lays a great platform for future applications in immunotherapy. Oral drug delivery is the most conventional and convenient administration route for the treatment of various diseases, as it alleviates the pain caused by injections with desirable cost-effectiveness and shows a good safety profile . However1/W/O2) and water-in-oil-in-water (W1/O/W2) are often used in the co-delivery of hydrophilic and hydrophobic bioactive compounds [Currently, emulsion technology has attracted much attention, as it has a low cost, involves a simple fabrication process with great stability, and can be easily scaled up in manufacturing; thus, it is widely applied in food, pharmaceutical, and nutraceutical products . Double-ompounds . Althougompounds . TherefoAt present, there has been increasing interest in the application of polysaccharides as polymeric biomaterials in drug delivery, since they are featured as biodegradable and biocompatible materials with controllable rigidity and functionality . Additio1/O/W2 emulsion to encapsulate a programmed death ligand-1 (PD-L1) blocking peptide and simvastatin. PD-L1 inhibitor is a major immune checkpoint inhibitor that is emerging as a front-line treatment for various types of cancer [2 (lower-case letters represent D-amino acids) and proved its excellent anti-tumor efficacy; thus, it was selected as a model peptide drug and encapsulated in the internal aqueous phase of a double emulsion [1/O/W2 double emulsions. In this study, the double emulsion was characterized, which included the droplet size and size distribution, zeta potential, encapsulation efficiency, morphology, viscosity, and drug release. In addition, the drug uptake in Caco-2 cells was also determined. Moreover, to better understand the role of the polysaccharide-modified double-phase emulsion, the immune-modulating effect of the dietary polysaccharides was also investigated for the first time for the application of an emulsion in oral delivery. Thereby, this research has developed a green, easily fabricated delivery system for the oral delivery of a peptide drug and a small-molecule drug with the aim of enhancing the drugs’ oral bioavailability and of demonstrating that dietary polysaccharides have great potential for use as carrier systems and immune-modulating agents.The objective of this research was to establish a green delivery system for a dietary-polysaccharide-modified fish-oil-based Wf cancer . Our resemulsion . Simvastemulsion . Thus, wFish oil  was purchased from Shanghai Yien Chemical Technology Co., Ltd. . Lecithin was purchased from Beijing Solarbio Technology Co., Ltd. . Okra polysaccharide was purchased from Rundekang Biotechnology Co., Ltd. . Yam polysaccharide and spirulina polysaccharide were purchased from Mixianer Biotechnology Co., Ltd. . Glycerin was purchased from Shanghai Aladdin Biochemical Co., Ltd. . All other reagents were of analytical grade. Murine mononuclear macrophages (RAW 264.7 cells) and the human colon adenocarcinoma cell line (Caco-2 cells) were conserved in our laboratory. RAW 264.7 cells and Caco-2 cells were cultured in DMEM . C57BL/6 female mice aged three to five weeks were maintained in a specific pathogen-free facility. Animal experimental procedures were carried out while following the national and institutional guidelines and were approved by the Ethics Committee of the Sun Yat-sen University.The standard solid-phase Fmoc synthesis method was used for peptide synthesis. Rink resin was used as a polymeric carrier. First, the resin was swelled and thoroughly washed. A hexahydropyridine/dimethyl formamide (DMF) mixture (1:4 or 1:1) was used to remove the protecting group of Fmoc from amino acids. After washing the resin with DMF, dichloromethane (DCM), the first amino acid, was added and attached to the resin by using a peptide condensation reagent, HCTU, and DIEA. The ninhydrin method was used to ensure the success of Fmoc removal and amino acid attachment. This process was repeated to obtain the initial peptide chain. Finally, the peptide was released from the resin by using trifluoroacetic acid, precipitated with ether, and centrifuged to obtain the crude peptide. Reverse-phase high-performance liquid chromatography (RP-HPLC) was used for peptide purification. Electrospray ionization mass spectrometry (ESI-MS) was used to verify the molecular weight of the peptide.2, in which PEG4 was used as a linker between FITC and the PD-L1 peptide. The synthesis of PEG4-Gqsehhmrvysf-NH2 was performed by using the standard solid-phase Fmoc synthesis method, as mentioned above. After deprotection, excess FITC was dissolved in 4 mL of DMF and added into the reaction tube with 800 μL DIEA, shaken, and allowed to react overnight in the dark. Finally, the processes of peptide release from the resin, precipitation, purification, and identification were carried out.Fluorescein labeling of the OPBP-1 peptide was performed according to the literature . The pep1), the oil phase (O), and the external aqueous phase (W2) were 1:3:12. Lecithin (1.5% w/v) was dissolved in fish oil to obtain the oil phase. Deionized water was added to the oil phase dropwise and stirred for 10 min, followed by a homogenization process for 3 min by using ultrasonic processor. The primary W1/O emulsion was obtained. Different polysaccharide solutions  with different concentrations  were prepared. Glycerin (8% v/v) was added to each solution to form an outer aqueous phase. The primary W1/O emulsion was added to the outer aqueous phase, and the dispersions were homogenized for 3 min to obtain the final W1/O/W2 double emulsions.The proportions of the inner aqueous phase  tubes and stored at 25 °C (room temperature) and 37 °C (body temperature) for 21 days. The stratification was observed, and 9 emulsions with poor stabilities were eliminated. As for the forced thermal stability study [Samples were placed on the glass slides and gently covered with a coverslip to prevent the emulsions from being destroyed. An optical microscope  was employed to observe the morphology of the emulsions at room temperature. A digital camera was used to acquire images.1/O/W2 emulsions. D was used to represent the average droplet size of the emulsions. The zeta potential [A Mastersizer 2000  was employed to determine the droplet size in the Wotential  was deteotential . The equD, D, and D are the volume size diameters at 10%, 50%, and 90% of the cumulative volume, respectively.2 phase) [g at 4 °C for 15 min, and they were separated into creamy layers  and a whey layer . The whey layer contained the unencapsulated drug and was collected from each centrifugal sample by using a syringe. This process was repeated 2–3 times in order to completely remove the creamy layers. The EE% of the W1/O/W2 emulsions was estimated by using the equation:free represents the amount of the drug penetrating into the external water phase (μg), and mtotal represents the total amount of the drug added to the emulsion (μg).The encapsulation efficiency (EE) of the drugs in the double emulsion was determined by measuring the concentrations of the drugs in the external aqueous phase (W2 phase) . The emu−1 at 25 °C [The viscosity of the emulsions was assessed by using a rheometer . Shear flow tests were carried out to study the viscosity with a shear rate ranging from 0.1 to 100 sat 25 °C .To evaluate the drug release profile, 5 mL of free drug solution or 5 mL of drug-loaded double emulsions were transferred into a dialysis bag  and dialyzed against 100 mL of simulated intestinal fluid (enzyme-free) at 37 °C with a stirring speed of 200 rpm/min. Samples were collected at specific time intervals. The concentration of the released drug was determined with a fluorescence microplate . The accumulative release of the drug was calculated.1/O/W2 emulsions were added at 10% (v/v), 5% (v/v), 2.5% (v/v), 1.25% (v/v), 0.625% (v/v), and 0.3125% (v/v) and incubated for 2, 4, 6, 12, and 24 h, respectively. MTT was added into each well. When the time point was reached, the medium was discarded, and DMSO was added to fully dissolve the purple crystals. The absorbance was read at the OD of 490 nm.Intestinal Caco-2 cells were seeded into 96-well plates one night in advance for cell adherence. The next day, the medium was discarded, and serum-free DMEM medium was added for cell starvation. After 8 h, the serum-free medium was discarded. WCaco-2 cells were seeded in laser confocal cell dishes one night in advance for cell adherence. A replacement with clean culture medium took place the next day. A total of 100 μL of 0.2 mg/mL free FITC-modified peptide or peptide-loaded double emulsion was added and incubated with the cells for 2 h at 37 °C Then, the dishes were rinsed with phosphate-buffered saline (PBS) to remove residual FITC-modified peptides. Hoechst 33,258 was added and incubated for 30 min at 37 °C for nuclear staining. PBS was used to remove residual Hoechst. Laser-scanning confocal microscopy (LSCM) was employed to observe the peptide uptake by Caco-2 cells.Briefly, Caco-2 cells were seeded into 48-well plate one night in advance for cell adherence. A replacement with fresh culture medium took place the next day. A total of 100 μL of 0.05 mg/mL free FITC-modified peptide or peptide-loaded double emulsion was added and incubated with the cells for 2 h at 37 °C. Then, the cells were collected and fixed, and the fluorescence intensity was measured via flow cytometry.5 cells/well were cultured, and 5 groups were set up: negative control of PBS, positive control of 1 μg/mL lipopolysaccharide (LPS), and 50 μg/mL of okra polysaccharide, yam polysaccharide, and spirulina polysaccharide. After 24 h of incubation, the cells were collected, stained with anti-CD11c-APC, anti-CD80-PerCP-efluorTM 710, and anti-CD86-PE, and analyzed via flow cytometry.This study was conducted by using bone-marrow-derived dendritic cells (BMDCs) from mice . Briefly5 cells/well, and the corresponding substances were added after starvation for 8 h. A total of 5 groups were set up: negative control of PBS, positive control of 1 μg/mL LPS, and 50 μg/mL of okra polysaccharide, yam polysaccharide, and spirulina polysaccharide. Over 24 h of incubation, the cells were lysed to extract RNA and reverse transcribed into cDNA, and the mRNA levels of IL-6 and TNF-α were detected via qPCR.Murine macrophages (RAW 264.7 cells) were cultured in a 24-well plate at a density of 2 × 10t-test for the differences between two groups. All data are shown as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.The statistical analysis was conducted with a one-tailed Student As shown in In the forced thermal stability studies at 45, 65, and 85 °C, the stratification of the emulsions was observed. As shown in The droplet sizes and zeta potentials of the emulsions were measured. As shown in −1, which was consistent with the characteristics of a Newtonian fluid. This result reflected the low viscosity of the emulsions, which could improve the mouthfeel during oral administration, reduce the adhesion to the esophageal mucosa, and be friendly to patients with dysphagia or children and the elderly.A flow curve is a graphical representation of how a flowing material behaves as the shear rate increases or decreases. The shape of the flow curve can distinguish the type of fluid. As shown in As shown in As shown in As shown in The effects of the polysaccharides on macrophages were also explored. Okra, yam, and spirulina polysaccharides significantly increased the IL-6 mRNA levels in RAW 264.7 cells. Okra and yam polysaccharides also increased the TNF-α mRNA levels . IL-6 ca1/O emulsion. The most stable formulations were obtained from a long-term storage stability study and a forced thermal stability study. The results showed that a lower concentration of the polysaccharide solution as the outer aqueous phase might be more conducive to the stability of the double emulsion. There might be a range of stability fluctuations between low-concentration polysaccharide solutions, which requires further investigation.This study constructed a green oral delivery system based on dietary food. Fish oil is a well-known nutrient that reduces the risk of cardiovascular diseases and diabetes, relieves arthritis, depression, and anxiety, and has immunomodulatory and anti-tumor effects . Dietary1/O/W2 double emulsions is their ability to simultaneously load a water-soluble drug and an oil-soluble drug within their internal aqueous phase and oil phase [The optimal emulsions of the three polysaccharides had similar characteristics in terms of their nano-size, which is advantageous for oral absorption compared with micro-size droplets . The emuil phase . The emuil phase . The resil phase .1/O/W2 emulsion was evaluated by using the technique of diffusion in a dialysis bag. The drug release profile was similar to that observed from double-phase emulsions reported in the literature, which is generally well correlated with pharmacokinetic results [The in vitro drug release from the W results . Irrespe results .One of the main purposes of this study was to maximize the oral bioavailability of PD-L1 blocking peptides and improve the anti-tumor effects through the aid of carriers. In addition, studies have shown that PD-1/PD-L1 blockade therapy combined with simvastatin has better anti-tumor effects. Therefore, we chose simvastatin as a lipophilic drug that was loaded into the oil phase. The optimal polysaccharide-modified emulsions had high drug encapsulation efficiencies, which were very similar to those reported in the literature . AdditioFinally, we evaluated the effects of okra, yam, and spirulina polysaccharides on the two major immune cells, dendritic cells and macrophages. More specifically, we investigated whether these polysaccharides could promote dendritic cell maturation and enhance cytokine expression in macrophages. The results indicated that okra and spirulina polysaccharides significantly increased the expression of CD80, CD86, and CD40 on the surface of BMDCs, indicating that these polysaccharides promoted dendritic cell maturation, which is conducive for antigen presentation and activation of naive T cells . In addiIn this study, dietary-polysaccharide-modified fish-oil-based double emulsions were successfully prepared through a simple emulsification method. The optimal emulsions were nano-sized and exhibited a negative surface charge, high drug encapsulation efficiency of over 97%, low viscosity, sustained drug release manner, and ability to improve oral drug absorption. In addition, these dietary polysaccharides could promote dendritic cell maturation and enhance cytokine expression in macrophages, which demonstrated that these dietary polysaccharides have dual roles of functioning as promising carrier systems and as potential immune modulators or adjuvants.